Jeong Seri, Kim Jung Ok, Jeong Seok Hoon, Bae Il Kwon, Song Wonkeun
Department of Laboratory Medicine, Kosin University College of Medicine, Busan, Republic of Korea.
Department of Laboratory Medicine, Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea.
J Microbiol Methods. 2015 Jun;113:4-9. doi: 10.1016/j.mimet.2015.03.019. Epub 2015 Mar 25.
The emergence of clinical isolates of carbapenemase-producing microbes confers multidrug-resistance to these bacteria and renders them difficult to treat. This study was performed to evaluate peptide nucleic acid (PNA) probe-based multiplex real-time PCR kits used to detect carbapenemase genes. In total, 324 carbapenemase genes, collected from 318 gram-negative clinical isolates in 36 different hospital laboratories, were assayed to evaluate multiplex real-time PCR kits (PANAGENE; Daejeon, Korea). The nine most prevalent carbapenemase genes (KPC, OXA-48, GES, IMP, VIM, NDM, ISAba1-OXA-51, OXA-23, and OXA-58) were included in this study. The sensitivity and specificity of the multiplex real-time PCR assay to all of the carbapenemase genes were above 99.0%, except for ISAba1-OXA-51. The detection limit of the assay was 100 target copies per 25 μL of reaction volume for all of the nine genetic types of carbapenemases, and the genes were all detected in a single three-hour PCR. The assay also showed considerable efficiency (above 80.0%), stable reproducibility (coefficient of variation, below 5.0%) and a long shelf-life (more than eight months) with no cross reactivity. The developed PNA-mediated multiplex real-time PCR assay was useful for the rapid, accurate and simultaneous identification of nine carbapenemase genes in gram-negative clinical isolates, suggesting its potential to help choose the appropriate antibiotics and aid the control of carbapenemase genes.
产碳青霉烯酶微生物临床分离株的出现使这些细菌具有多重耐药性，难以治疗。本研究旨在评估用于检测碳青霉烯酶基因的基于肽核酸（PNA）探针的多重实时PCR试剂盒。总共对从36个不同医院实验室的318株革兰氏阴性临床分离株中收集的324个碳青霉烯酶基因进行检测，以评估多重实时PCR试剂盒（PANAGENE；韩国大田）。本研究纳入了9种最常见的碳青霉烯酶基因（KPC、OXA-48、GES、IMP、VIM、NDM、ISAba1-OXA-51、OXA-23和OXA-58）。除ISAba1-OXA-51外，多重实时PCR检测对所有碳青霉烯酶基因的敏感性和特异性均高于99.0%。该检测方法对所有9种碳青霉烯酶基因类型的检测限为每25μL反应体积100个靶标拷贝，并且所有基因均可在单次三小时PCR中检测到。该检测方法还显示出相当高的效率（高于80.0%）、稳定的重复性（变异系数低于5.0%）和较长的保质期（超过8个月），且无交叉反应。所开发的PNA介导的多重实时PCR检测方法可用于快速、准确且同时鉴定革兰氏阴性临床分离株中的9种碳青霉烯酶基因，表明其有助于选择合适的抗生素以及辅助控制碳青霉烯酶基因的潜力。
